CN115433087A - A method for extracting dibutyl terephthalate from Raffaelea lauricola - Google Patents

Abstract

The invention discloses a method for extracting dibutyl terephthalate from Raffaelea lauricola , comprising: activating bacterial strains, primary culture, secondary culture, bacterial body grinding, extraction with ethyl acetate, concentration to obtain a crude extract, and the crude extract The extract was subjected to reverse-phase silica gel column chromatography, eluted with methanol:water gradient, and then purified by multiple 200-300 mesh silica gel column chromatography, and the eluate was collected and concentrated to obtain the target product. ¹ H NMR, ¹³ C NMR, COZY, HMBC, HSQC spectrogram analysis proved that dibutyl terephthalate was extracted from the metabolites of the bacteria for the first time.

Description

A kind of extracting dibutyl terephthalate from Raffaelea lauricola method

technical field

The invention belongs to the technical field of separating active substances from microorganisms, and in particular relates to a method for extracting dibutyl terephthalate (1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate) from Raffaelea lauricola .

Background technique

Raffaelea lauricola is a highly pathogenic companion fungus carried by Ambrosia beetle , and it is a pathogenic fungus that causes tree wilt. People have been studying its pathogenic mechanism. With the deepening of research, other strains of this genus Some active substances such as inhibiting plant growth and antibacterial have been isolated from the plant. This indicates that the bacterium has great application value in biopesticide or medicine.

Currently, there is no report on the isolation of 1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate from Raffaelea lauricola .

Contents of the invention

The purpose of this invention is to provide a method for extracting dibutyl terephthalate (1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate) from Raffaelea lauricola , to promote its further research and development in the field of medicine develop.

In order to achieve the above object, the technical scheme that the present invention takes is as follows:

A method for extracting dibutyl terephthalate (1-Butyl4-(2-methylpropyl) 1,4-benzonedicarboxylate) from Raffaelea lauricola , comprising the following steps:

1) After activating Raffaelea lauricola , take the bacterial block and inoculate it into the PDB medium, and culture it continuously for 5-10 days under the conditions of 140-180r/min and 20-28°C, and transfer it according to the inoculation amount of 15wt%~20wt% Put it into the improved rice culture medium, continue static culture at 20-28° C. for 28-40 days, and obtain mycelia.

2) Dry the mycelia obtained in step 1) at room temperature, grind and pulverize, soak in 1-2 times the volume of ethyl acetate for 12-24 hours, ultrasonicate for 40-60 minutes, collect the extract, and add bacteria to the residue Ethyl acetate with 1-2 times the volume of silk, ultrasonic for 40-60 minutes, collect the extract, repeat the above steps to obtain a total of three extracts, combine and concentrate to obtain the crude extract extract;

3) Dissolve the extract in chloroform or dichloromethane, then mix the sample with reversed-phase silica gel C ₁₈ , dry-pack the column, elute with methanol:water gradient from volume ratio 1:9-9:1, and collect methanol: The water volume ratio is 9:1, and part of the eluate is concentrated by rotary evaporation to obtain component A.

4) Dissolve component A with 1 times the volume of chloroform, pass through a 200-300 mesh silica gel column, start gradient elution with a volume ratio of petroleum ether:ethyl acetate of 30:1, and collect petroleum ether:ethyl acetate volume ratio of 10: 1 Part of the eluate was concentrated by rotary evaporation to obtain extract, then passed through a 200-300 mesh silica gel column, and started gradient elution with a volume ratio of petroleum ether: chloroform of 8:1, and collected the part with a volume ratio of petroleum ether: chloroform of 2:1 The eluate was suspended and evaporated to obtain white powder 1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate, whose structural formula is as follows:

。

.

Further, the improved rice culture medium in step 1): 100 g of rice, 10 g of wood chips, 120 mL of pure water, and autoclave at 121° C. for 20 minutes for later use.

Further, the size of the inoculum block in the step 1) is 0.5×0.5 cm, and the inoculum size is 4 blocks.

Further, the power of the ultrasound in the step 2) is 400 W, and the frequency is 35 KHz.

Further, the reversed-phase silica gel C ₁₈ used in step 3) has a particle size of 40-60 μm and a pore size of 120 Å.

Further, the gradient elution ratio in step 3) is methanol: water volume ratio 1:9, 2:8, 3:7, 4:6, 6:4, 7:3, 8:2, 9:1 .

Further, in the step 4) gradient elution, the volume ratios of petroleum ether: ethyl acetate are 30:1, 20:1, 10:1, 8:1, 6:1, 3:1, petroleum ether: chloroform The volume ratios are 8:1, 6:1, 4:1, 2:1 in turn.

Significant advantage of the present invention:

The separation and purification method of the invention is simple, low in cost and easy to operate; and the prepared 1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate has high purity and good repeatability.

Description of drawings

Figure 1 is the ¹ HNMR spectrum (CDCl ₃ ) of 1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate compound;

Figure 2 is the ¹³ CNMR spectrum (CDCl ₃ ) of 1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate compound;

Figure 3 is the DEPT135 spectrum (CDCl ₃ ) of 1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate compound;

Figure 4 is the COZY spectrum (CDCl ₃ ) of 1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate compound;

Figure 5 is the HMBC spectrum (CDCl ₃ ) of 1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate compound;

Figure 6 is the HSQC spectrum (CDCl ₃ ) of 1-Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate compound.

detailed description

In order to make the above-mentioned features and advantages of the present invention more comprehensible, specific embodiments are given below for detailed description. The methods of the present invention are conventional methods in the art unless otherwise specified.

The strain number of Raffaelea lauricola used in the present invention is Hulcr7161.

Example 1

1) Take Raffaelea lauricola as the material, pick 4 pieces of bacterial blocks with a size of 0.5×0.5 cm under sterile conditions and inoculate them into PDB medium (3.9 g of potato dextrose powder, 100 mL of pure water, put them into a 250 mL Erlenmeyer flask, Sterilized under high pressure at 121°C for 20 min), cultured continuously at 160 r/min and 25°C for 7 days, namely primary culture, and transferred to improved rice medium (rice 100 g, sawdust 10 g, add 120 mL of pure water, autoclave at 121°C for 20 minutes), and continue to culture at 25°C for 35 days, that is, secondary culture.

2) Collect the mycelium obtained after 35 days of cultivation, dry it at room temperature, grind and pulverize it, add 1 times the volume of ethyl acetate to soak for 12 hours, ultrasonicate for 40 minutes, filter to obtain the extract, add 1 times the mycelium to the residue to extract and ethyl acetate, ultrasonic for 40 minutes, the extract was collected, and the above steps were repeated again to obtain a total of three extracts, which were combined and concentrated to obtain the crude extract extract.

3) Dissolve the extract in chloroform, mix the sample with 40-60 μm particle size reversed-phase C ₁₈ silica gel, dry-pack the column, and start gradient elution with methanol:water from a volume ratio of 1:9 (the volume ratio of methanol:water is 1 :9, 2:8, 3:7, 4:6, 6:4, 7:3, 8:2, 9:1), collected methanol: water volume ratio 9:1 partial rotary evaporation to obtain the crude product.

4) Dissolve the crude product with 1 volume of chloroform, put it on a 200-300 mesh silica gel column, start with a volume ratio of petroleum ether: ethyl acetate of 30:1 (gradient elution ratios are 30:1, 20:1, 10:1) Gradient elution, collecting petroleum ether: ethyl acetate volume ratio of 10:1 part of the eluate, concentrated by rotary evaporation to obtain extract, and then passing through a 200-300 mesh silica gel column, starting with petroleum ether: chloroform volume ratio of 8:1 Gradient elution (gradient elution ratios are 8:1, 6:1, 4:1, 2:1), collect a part of the eluate with a volume ratio of petroleum ether: chloroform of 2:1, and suspend and evaporate to obtain a white powder 1- Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate.

In this embodiment, the ultrasonic power in step 2) is 400 W, and the frequency is 35 KHz;

The calculated extraction rate is 0.67% (extraction rate%=weight/total amount of mycelium extract×100%).

Example 2

1) Take Raffaelea lauricola as the material, pick 4 pieces of bacteria with a size of 0.5×0.5 cm under aseptic conditions and inoculate them into PDB medium (3.9 g of potato dextrose powder, 100 mL of pure water, put them into a 250 mL Erlenmeyer flask, Sterilized under high pressure at 121°C for 20 min), cultured continuously at 140 r/min and 20°C for 5 days, namely primary culture, and transferred to improved rice medium (rice 100 g, sawdust 10 g, add 120 mL of pure water, autoclave at 121°C for 20 min), and continue to culture at 20°C for 25 days, which is the secondary culture.

2) Collect and cultivate the mycelium for 25 days, dry it at room temperature, grind and pulverize it, add 1 volume of ethyl acetate to soak for 12 hours, ultrasonicate for 40 minutes, filter to obtain the extract, add 1 volume of mycelium to the residue of acetic acid Ethyl ester, sonicated for 40 minutes, the extract was collected, and the above steps were repeated again to obtain a total of three extracts, which were combined and concentrated to obtain the crude extract extract.

3) Dissolve the extract in chloroform, mix the sample with reversed-phase C ₁₈ silica gel with a particle size of 40-60 μm, carry out crude fractionation under reduced pressure, and start gradient elution with methanol:water starting from a volume ratio of 1:9 (methanol:water ratio is 1:9, 2:8, 3:7, 4:6, 6:4, 7:3, 8:2, 9:1), and the crude product obtained by partial rotary evaporation of methanol:water volume ratio of 9:1 was collected.

4) Dissolve the crude product with 1 volume of chloroform, put it on a 200-300 mesh silica gel column, start gradient elution with a volume ratio of petroleum ether:ethyl acetate of 30:1 (gradient elution ratios are 30:1, 20:1, 10 :1), collect petroleum ether: ethyl acetate volume ratio of 10:1 part of the eluate, concentrate by rotary evaporation to obtain extract, then pass through a 200-300 mesh silica gel column, and start gradient elution with petroleum ether: chloroform 8:1 (Gradient elution ratios are 8:1, 6:1, 4:1, 2:1), collect petroleum ether: chloroform volume ratio of 2:1 part of the eluate, rotary evaporation to obtain white powder 1-Butyl 4-( 2-methylpropyl) 1,4-benzonedicarboxylate.

In this embodiment, the ultrasonic power in step 2) is 400 W, and the frequency is 35 KHz;

The calculated extraction rate is 0.72% (extraction rate%=weight/total amount of mycelium extract×100%).

Example 3

1) Take Raffaelea lauricola as the material, pick 4 pieces of bacterial blocks with a size of 0.5×0.5 cm under sterile conditions and inoculate them into PDB medium (3.9 g of potato glucose powder, 100 mL of pure water, put them into a 250 mL Erlenmeyer flask, Sterilized under high pressure at 121°C for 20min), cultured continuously at 180 r/min and 28°C for 10 days, namely primary culture, and transferred to improved rice medium (rice 100 g, sawdust 10 g , add 120 mL of pure water, autoclave at 121°C for 20 min), and continue to culture at 28°C for 40 days, that is, secondary culture.

2) Collect and cultivate the mycelium for 40 days, dry it at room temperature, grind it, add 1 volume of ethyl acetate to soak for 12 hours, ultrasonicate for 90 minutes, filter to obtain the extract, add 1 volume of acetic acid to the residue Ethyl ester, sonicated for 90 minutes, the extract was collected, and the above steps were repeated again to obtain a total of three extracts, which were combined and concentrated to obtain the crude extract extract.

3) Dissolve the extract in chloroform, mix the sample with reversed-phase C ₁₈ silica gel with a particle size of 40-60 μm, dry-pack the column, and start gradient elution with methanol:water from a volume ratio of 1:9 (the ratio of methanol:water is 1 :9, 2:8, 3:7, 4:6, 6:4, 7:3, 8:2, 9:1), collect methanol: water volume ratio of 9:1 and partially rotary evaporate the crude product.

4) Dissolve the crude product with 1 volume of chloroform, put it on a 200-300 mesh silica gel column, start gradient elution with a volume ratio of petroleum ether:ethyl acetate of 30:1 (gradient elution ratios are 30:1, 20:1, 10 :1), collect petroleum ether: ethyl acetate volume ratio of 10:1 part of the eluate, concentrate by rotary evaporation to obtain extract, and then pass through a 200-300 mesh silica gel column, starting with petroleum ether: chloroform volume ratio of 8:1 Gradient elution (gradient elution ratios are 8:1, 6:1, 4:1, 2:1), collect a part of the eluate with a volume ratio of petroleum ether: chloroform of 2:1, and suspend and evaporate to obtain a white powder 1- Butyl 4-(2-methylpropyl) 1,4-benzonedicarboxylate.

In this embodiment, the ultrasonic power in step 2) is 400 W, and the frequency is 35 KHz;

The calculated extraction rate is 0.74% (extraction rate%=weight/total amount of mycelium extract×100%).

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be determined by the claims.

Claims (7)

1. a method for extracting dibutyl terephthalate from Raffaelea lauricola , is characterized in that, comprises the following steps: 1) After activating Raffaelea lauricola , take the bacterial block and inoculate it into the PDB medium, culture continuously for 5-10 days at 160-180 r/min, 20-28°C, and transfer to In the rice culture medium, continue to culture statically at 20-28°C for 28-40 days to obtain mycelium; 2) Dry the mycelium obtained in step 1) at 35~40°C and grind it into a powder, soak it in 1~2 times the volume of ethyl acetate for 12~24 hours, ultrasonicate for 40~60 minutes, collect the extract and residue Then add ethyl acetate of 1 to 2 times the volume of mycelia, ultrasonic for 40 to 60 minutes, collect the extract, repeat the above steps, and obtain a total of three extracts that are combined and concentrated to obtain a crude extract extract; 3) Dissolve the crude extract extract in chloroform or dichloromethane, then mix the sample with reversed-phase silica gel C ₁₈ , dry-pack the column, and use methanol:water volume ratio of 1:9-9:1 gradient elution, Collect methanol: water with a volume ratio of 9:1 part of the eluate, and concentrate by rotary evaporation to obtain component A; 4) Dissolve component A with 1 times the volume of chloroform, pass through a 200-300 mesh silica gel column, start gradient elution with a volume ratio of petroleum ether:ethyl acetate of 30:1, and collect petroleum ether:ethyl acetate volume ratio of 10: 1 Part of the eluate was concentrated by rotary evaporation to obtain extract, then passed through a 200-300 mesh silica gel column, and started gradient elution with a volume ratio of petroleum ether: chloroform of 8:1, and collected the part with a volume ratio of petroleum ether: chloroform of 2:1 The eluent was evaporated by rotary evaporation to obtain a white powder, namely dibutyl terephthalate. 2. The method for extracting dibutyl terephthalate from Raffaelea lauricola according to claim 1, characterized in that: the size of the bacterial block in step 1) is 0.5×0.5 cm, and the inoculum size is 4 blocks. 3. The method for extracting dibutyl terephthalate from Raffaelea lauricola according to claim 1, characterized in that: the rice medium in step 1) consists of: 100 g of rice, 10 g of sawdust, and 120 g of pure water mL; use after autoclaving at 121°C for 20 min. 4. The method for extracting dibutyl terephthalate from Raffaelea lauricola according to claim 1, characterized in that: in step 2), the ultrasonic power is 400 W and the frequency is 35 KHz. 5. The method for extracting dibutyl terephthalate from Raffaelea lauricola according to claim 1, characterized in that: the reversed-phase silica gel C ₁₈ used in step 3) has a particle size of 40-60 μm and a pore size of 120 Å. 6. A method for extracting hopane-type triterpenoids from Raffaelea lauricola according to claim 1, characterized in that: the gradient elution ratio in step 3) is methanol: water volume ratio is 1:9 , 2:8, 3:7, 4:6,6:4, 7:3, 8:2, 9:1. 7. The method for extracting dibutyl terephthalate from Raffaelea lauricola according to claim 1, characterized in that: in the gradient elution of step 4), the volume ratio of petroleum ether: ethyl acetate is 30:1, 20 :1, 10:1, 8:1,6:1, 3:1; the volume ratio of petroleum ether: chloroform is 8:1, 6:1, 4:1, 2:1 in sequence.

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